Compositions comprising polynucleotides encoding RDCVF1 or RDCVF2

ABSTRACT

Disclosed are methods and compositions for early diagnosis, monitoring and treatment of retinal dystrophy, age-related macular degeneration, Bardet-Biedel syndrome, Bassen-kornzweig syndrome, best disease, choroidema, gyrate atrophy, congenital amourosis, refsun syndrome, stargardt disease and Usher syndrome. In particular, the invention relates to a protein, termed “Rdcvf1,” that is differentially transcribed and expressed in subjects suffering from retinal dystrophies and the like, such as retinal dystrophy and age-related macular degeneration compared with nonsufferers, antibodies which recognize this protein, and methods for diagnosing such conditions.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/739,739, filed Apr. 25, 2007, now U.S. Pat. No. 8,114,849,which is a divisional of U.S. patent application Ser. No. 10/473,008,filed Apr. 2, 2004, now U.S. Pat. No. 7,795,387, which claims thebenefit of and which is a national stage filing of InternationalApplication No. PCT/EP2002/003810, filed on Apr. 5, 2002, which claimspriority to, and the benefit of, French Patent Application No. 0104712,filed Apr. 6, 2001, the contents of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to methods and compositions for detection andtreatment of retinal degenerative diseases. In particular, the inventionrelates to a protein that protects against cone degeneration, nucleicacid molecules that encode such a protein, antibodies that recognize theprotein, and methods for diagnosing retinal degenerative diseases.

BACKGROUND OF THE INVENTION

Photoreceptors are a specialized subset of retinal neurons, that areresponsible for vision. Photoreceptors consist of rods and cones whichare the photosensitive cells of the retina. Each rod and cone elaboratesa specialized cilium, referred to as an outer segment, that houses thephototransduction machinery. The rods contain a specific light-absorbingvisual pigment, rhodopsin. There are three classes of cones in humans,characterized by the expression of distinct visual pigments: the bluecone, green cone and red cone pigments. Each type of visual pigmentprotein is tuned to absorb light maximally at different wavelengths. Therod rhodopsin mediates scotopic vision (in dim light), whereas the conepigments are responsible for photopic vision (in bright light). The red,blue and green pigments also form the basis of color vision in humans.The visual pigments in rods and cones respond to light and generate anaction potential in the output cells, the rod bipolar neurons, which isthen relayed by the retinal ganglion neurons to produce a visualstimulus in the visual cortex.

In humans, a number of diseases of the retina involve the progressivedegeneration and eventual death of photoreceptors, leading inexorably toblindness. Degeneration of photoreceptors, such as by inherited retinaldystrophies (e.g., retinitis pigmentosa), agerelated maculardegeneration and other maculopathies, or retinal detachment, are allcharacterized by the progressive atrophy and loss of function ofphotoreceptor outer segments. In addition, death of photoreceptors orloss of photoreceptor function results in partial deafferentation ofsecond order retinal neurons (rod bipolar cells and horizontal cells) inpatients with retinal dystrophies, thereby decreasing the overallefficiency of the propagation of the electrical signal generated byphotoreceptors. Secondary glial and pigment epithelium changes secondaryto photoreceptors degeneration result in vascular changes leading toischemia and gliosis. Trophic factors that are capable of rescuingphotoreceptors from cell death and/or restoring the function ofdysfunctional (atrophic or dystrophic) photoreceptors may representuseful therapies for the treatment of such conditions.

The progression of these conditions points to a sequential loss of thetwo classes of photoreceptors: initially rods are lost as a directresult of a genetic or environmental or unknown lesion, resulting innight blindness and a reduction in visual field followed inevitably byloss of cones leading to total blindness. Thus, cones die indirectlysince they do not express the primary lesion.

Not all of the genes associated with retinal dystrophy have yet beenidentified. Identification of such genes would make possible to bothdiagnose the disease and identify effective therapies.

SUMMARY OF THE INVENTION

The invention relates generally to a novel gene family, Rod-derived ConeViability Factor (Rdcvf). In a first aspect, the invention provides anisolated polypeptide with an amino acid sequence as set forth in SEQ IDNO:2 or SEQ ID NO:4. Such a polypeptide, or fragments thereof, is foundin the eye of sufferers of retinal dystrophies to a much lesser extentthan in the eye of individuals without retinal dystrophy. Fragments ofthe isolated polypeptide with an amino acid sequence as set forth in SEQID NO:2 or SEQ ID NO:4 will comprise polypeptides comprising from about5 to 10 amino acids, preferably from about 10 to about 20 amino acids,more preferably from about 20 to about 100 amino acids, and mostpreferably from about 20 to about 50 amino acids. In accordance withthis aspect of the invention there are provided a novel polypeptide ofmammalian origin, and in particular of mouse or human origin as well asbiologically, diagnostically or therapeutically useful fragments,variants and derivatives thereof, variants and derivatives of thefragments, and analogs of the foregoing. Also within the scope of thepresent invention are polypeptides that are substantially similar to thepolypeptide with the amino acid sequence as set forth in SEQ ID NO:2 orSEQ ID NO:4 e.g. an amino acid sequence as set forth in SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.

In a second aspect, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence as set forth in SEQ ID NO:1 orSEQ ID NO:3. Also within the scope of the present invention are nucleicacids that are substantially similar to the nucleic acid with thenucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3 e.g.nucleotide sequences as set forth in SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. In a preferred embodiment, theinvention provides an isolated nucleic acid molecule that encodes for apolypeptide selected from the group consisting of the polypeptides setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12 and SEQ ID NO:14, e.g. nucleotides 45-374 of SEQ IDNO:1, nucleotides 26-676 of SEQ ID NO:3, nucleotides 24-353 of SEQ IDNO:5, nucleotides 48-686 of SEQ ID NO:7, nucleotides 265-570 of SEQ IDNO:9, nucleotides 300-770 of SEQ ID NO: 11 or nucleotides 331-738 of SEQID NO:13. In a preferred embodiment, the isolated DNA takes the form ofa vector molecule comprising the DNA as set forth in SEQ ID NO:1 or SEQID NO:3.

A third aspect of the present invention encompasses a method for thediagnosis of retinal dystrophy in a human which includes detecting thedecreased transcription of messenger RNA transcribed from Rdcvf1 orRdcvf2-encoding DNA in the eye from a mammalian organism, preferably ahuman, where such decreased transcription is diagnostic of theorganisms' affliction with retinal dystrophy or pathological aging(ARMD). Another embodiment of the assay aspect of the invention providesa method for the diagnosis of retinal dystrophy in a mammalian organism,preferably a human, which requires measuring the amount of a Rdcvf1 orRdcvf2 polypeptide or fragments thereof in the eye of a human suspectedof suffering form a retinal dystrophy, where the presence of a decreasedamount of the polypeptide or fragments thereof, relative to the amountof the polypeptide or fragments thereof in the eye of an individual notsuffering from a retinal dystrophy, is diagnostic of the human'ssuffering from retinal dystrophy.

In accordance with another aspect of the invention there are providedanti-sense polynucleotides that regulate transcription of the Rdcvf1 orRdcvf2 gene; in another embodiment, double stranded RNA is provided thatcan regulate the transcription of the Rdcvf1 or Rdcvf2 gene.

Another aspect of the invention provides a process for producing theaforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing. In a preferred embodiment of this aspect of the inventionthere are provided methods for producing the aforementioned Rdcvf1polypeptides comprising culturing host cells having incorporated thereinan expression vector containing an exogenously-derived Rdcvf1 orRdcvf2-encoding polynucleotide under conditions sufficient forexpression of Rdcvf1 or Rdcvf2 polypeptides in the host and thenrecovering the expressed polypeptide.

In accordance with another aspect of the invention there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for, inter alia,research, biological, clinical and therapeutic purposes.

In certain additional preferred aspects of the invention there areprovided an antibody or a fragment thereof which specifically binds to apolypeptide that comprises the amino acid sequence set forth in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8, i.e., Rdcvf1, or SEQ IDNO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, i.e. Rdcvf2. In certainparticularly preferred aspects in this regard, the antibodies are highlyselective for mammalian, preferably mouse and in particular human Rdcvf1or Rdcvf2 polypeptides or portions of such Rdcvf1 or Rdcvf2polypeptides. In a related aspect, an antibody or fragment thereof isprovided that binds to a fragment or portion of the amino acid sequenceset forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12 or SEQ ID NO:14.

In another aspect, methods of treating a disease in a subject, where thedisease is mediated by or associated with a change in Rdcvf1 or Rdcvf2gene expression e.g. a decrease in the presence of RDCVF1 or RDCVF2polypeptide in the eye, by the administration of a therapeuticallyeffective amount of a RDCVF1 or RDCVF2 protein as set out in SEQ ID NO:2SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQID NO:14 or a related protein or a fragment or portion thereof to thesubject is provided. Also provided are methods for the diagnosis of adisease or condition associated with an decrease in Rdcvf1 or Rdcvf2gene expression or decrease in the presence of RDCVF1 or RDCVF2polypeptide in a subject, which comprises utilizing an antibody thatbinds to a polypeptide with the amino acid sequence set out in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12or SEQ ID NO:14, or a fragment or portion thereof in an immunoassay.

In yet another aspect, the invention provides cells which can bepropagated in vitro, preferably vertebrate cells, which are capable upongrowth in culture of producing a polypeptide that comprises the aminoacid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14 or fragments thereof,where the cells contain transcriptional control DNA sequences, otherthan mouse or human Rdcvf1 or Rdcvf2 transcriptional control sequences,where the transcriptional control sequences control transcription of DNAencoding a polypeptide with the amino acid sequence according to SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12or SEQ ID NO:14 or fragments thereof.

In a related aspect, the present invention provides a method forproducing Rdcvf1 or Rdcvf2 polypeptides which comprises culturing a hostcell having incorporated therein an expression vector containing anexogenously-derived Rdcvf1 or Rdcvf2-encoding polynucleotide underconditions sufficient for expression of Rdcvf1 or Rdcvf2 polypeptides inthe host cell, thereby causing the production of an expressedpolypeptide, and recovering the expressed polypeptide.

In yet another aspect of the present invention there are provided assaymethods and kits comprising the components necessary to detectabnotinal, e.g. below-normal expression of Rdcvf1 or Rdcvf2polynucleotides or polypeptides or fragments thereof in body tissuesamples derived from a patient, such kits comprising e.g., antibodiesthat bind to Rdcvf1 or Rdcvf2 or oligonucleotide probes that hybridizewith polynucleotides of the invention. In a preferred embodiment, suchkits also comprise instructions detailing the procedures by which thekit components are to be used.

In another aspect, the invention is directed to an Rdcvf1 or Rdcvf2polypeptide for use in the treatment of a human or animal body. Arelated aspect is directed to the use of an Rdcvf1 or Rdcvf2 polypeptideor fragment thereof, nucleotide encoding Rdcvf1 or Rdcvf2 or a fragmentthereof, or antibody that binds to Rdcvf1 or Rdcvf2 or a fragmentthereof in the manufacture of a medicament to treat a retinal dystrophy.

In another aspect, the invention provides a retinoprotective agentcomprising a polypeptide selected from the group of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ IDNO:14, and, optionally, a pharmaceutically acceptable carrier. In arelated aspect the invention provides a pharmaceutical compositionscomprising a Rdcvf1 or Rdcvf2 polypeptide or fragment thereof,nucleotide encoding Rdcvf1 or Rdcvf2 or a fragment thereof, for thetreatment of a retinal dystrophy. In another related aspect, theinvention provides a pharmaceutical composition comprising atherapeutically effective amount of a polypeptide selected from thegroup of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12 or SEQ ID NO:14 and a pharmaceutically acceptablecarrier.

In a related aspect, the invention provides a method for the treatmentof retinal dystrophy comprising administering a therapeuticallyeffective amount of a polypeptide selected from the group of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12or SEQ ID NO:14, and pharmaceutically acceptable carrier, to a subjectin need.

In another aspect, the invention is directed to methods for theidentification of molecules that can bind to Rdcvf1 or Rdcvf2 and/ormodulate the activity of Rdcvf1 or Rdcvf2 or molecules that can bind tonucleic acid sequences that modulate the transcription or translation ofRdcvf1 or Rdcvf2. Such methods are disclosed in, e.g., U.S. Pat. Nos.5,541,070; 5,567,317; 5,593,853; 5,670,326; 5,679,582; 5,856,083;5,858,657; 5,866,341; 5,876,946; 5,989,814; 6,010,861; 6,020,141;6,030,779; and 6,043,024, all of which are incorporated by referenceherein in their entirety. Molecules identified by such methods also fallwithin the scope of the present invention.

In yet another aspect, the invention is directed to methods for theintroduction of nucleic acids of the invention into one or more tissuesof a subject in need of treatment with the result that one or moreproteins encoded by the nucleic acids are expressed and or secreted bycells within the tissue.

In another aspect the invention provides a method of providingphotoreceptor cells for implantation wherein the photoreceptor cells arecultured together with RdCVF1 or RdCVF2.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill from the following description.It should be understood, however, that the following description and thespecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only. Various changes andmodifications within the spirit and scope of the disclosed inventionwill become readily apparent to those skilled in the art from readingthe following description and from reading the other parts of thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: mouse Rdcvf1 nucleotide sequence from the expression cloning andmouse RdCVF1 amino acid sequence.

FIG. 2: mouse Rdcyf1L nucleotide sequence, and amino acid sequence.

FIG. 3: human Rdcvf1 and human Rdcvf1 amino acids sequence.

FIG. 4: human Rdcyf1L nucleotide sequence, human RdcyfIL amino acidssequence.

FIG. 5: mouse Rdcvf2 nucleotide sequence and: mouse Rdcvf2 amino acids.

FIG. 6: mouse Rdcvf2L nucleotide sequence and mouse Rdcvf2L amino acid.

FIG. 7: human Rdcvf2 nucleotide sequence, and human Rdcvf2 amino acidssequence.

FIG. 8: depicts an amino acid alignments of the short forms of Rdcvf:(SEQ ID No 2, 6, 10 and 14) and of the long forms of Rdcvf: SEQ ID No4,8, 12 and 14).

FIG. 9: depicts the primers for GST-Rdcvf1.

FIG. 10: Multiple Alignment of RDCVF1 (SEQ ID NO: 2)/RDCVF2 (SEQ ID NO:10).

FIG. 11: Comparison of mouse (SEQ ID NO: 10) and human (SEQ ID NO: 36)RDCVF2

FIG. 12: Multiple Alignment of mouse Rdcvf2 (SEQ ID NO: 37) with ESTclones be552141 (SEQ ID NO: 20), bi517442 (SEQ ID NO: 21), bg707818 (SEQID NO: 22) and bi603812 (SEQ ID NO: 23).

FIG. 13: Multiple Alignment of Rdcvf1 (SEQ ID NO: 38) with EST clonesbg299078 (SEQ ID NO: 15), ai716631 (SEQ ID NO: 16), bg294111 (SEQ ID NO:17), be108041 (SEQ ID NO: 18) and bg395178 (SEQ ID NO: 19).

FIG. 14: EST sequence bg299078 (SEQ ID NO: 15) corrected to matchRdcvf1.

FIG. 15: EST sequence bg294111 (SEQ ID NO: 17) corrected to matchRdcvf1L.

FIG. 16: Real-time RT-PCR analysis of the expression of rod arrestin (A)and RdCSF1 (B) in 5 weeks retina C57BL/6@N 5 weeks (gray) and C3H/HE@N(black).

FIG. 17: RT-PCR analysis showing that Rdcvf2 is expressed in arod-dependent manner and is expressed in another part of the CNS.

FIG. 18: PCR analysis showing that RdCVF1 is expressed in arod-dependent manner.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited hereinare hereby incorporated by reference in their entirety.

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA are used. Thesetechniques are well known and are explained in, for example, CurrentProtocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M.Ausubel ed.); Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II, 1985(D. N. Glover ed.); Oligonucleotide Synthesis, 1984 (M. L. Gait ed.);Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription andTranslation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986(R. I. Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press);Perbal, 1984, A Practical Guide to Molecular Cloning; the series,Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors forMammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold SpringHarbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wuand Grossman, and Wu, eds., respectively).

As used herein, “differentially expressed gene” refers to (a) a genecontaining at least one of the DNA sequences disclosed herein (e.g., asshown in FIG. 1 and SEQ ID NO:1 or as shown in FIG. 2 and SEQ ID NO:3);(b) any DNA sequence that encodes the amino acid sequence encoded by theDNA sequences disclosed herein (e.g., as shown in FIG. 1 and SEQ ID NO:2or as shown in FIG. 2 and SEQ ID NO:4); or (c) any DNA sequence that issubstantially similar to the coding sequences disclosed herein.

In its broadest sense, the term “substantially similar”, when usedherein with respect to a nucleotide sequence, means a nucleotidesequence corresponding to a reference nucleotide sequence, wherein thecorresponding sequence encodes a polypeptide having substantially thesame structure and function as the polypeptide encoded by the referencenucleotide sequence, e.g. Where only changes in amino acids notaffecting the polypeptide function occur. Desirably the substantiallysimilar nucleotide sequence encodes the polypeptide encoded by thereference nucleotide sequence. The percentage of identity between thesubstantially similar nucleotide sequence and the reference nucleotidesequence desirably is at least 90%, more preferably at least 95%, stillmore preferably at least 99%. Sequence comparisons are carried out usinga Smith-Waterman sequence alignment algorithm (see e.g. Waterman, M. S.Introduction to Computational Biology Maps, sequences and genomes.Chapman & Hall. London: 1995. ISBN 0-412-99391-0. The localS program,version 1.16, is used with the following parameters: match: 1, mismatchpenalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2. Anucleotide sequence “substantially similar” to the reference nucleotidesequence hybridizes to the reference nucleotide sequence in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in2×SSC, 0.1% SDS at 50° C., more desirably in 7% sodium dodecyl sulfate(SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDSat 50° C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50°C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., morepreferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 0.1×SSC, 0.1% SDS at 65° C., yet still encodes afunctionally equivalent gene product.

The differentially expressed genes disclosed herein are expressed in eyetissue and in particular is produced in rod cells however, in a humanafflicted with a retinal dystrophy such as retinitus pigmentosa,age-related macular degeneration, Bardet-Biedel syndrome,Bassen-kornzweig syndrome, best disease, choroidema, gyrate atrophy,congenital amourosis, refsun syndrome, stargardt disease and Ushersyndrome it is produced in decreases amounts relative to, i.e., to alesser extent than in the corresponding tissues of humans who do notsuffer from retinal dystrophy. Messenger RNA transcribed from thedifferentially expressed genes, and protein translated from such mRNA,is present in rod tissues and/or associated with such tissues in anamount at least about half, preferably at least about five times, morepreferably at least amount ten times, most preferably at least about 100times less than the levels of mRNA and protein found in correspondingtissues found in humans who do not suffer from a retinal dystrophy. Suchdecreases transcription of Rdcvf1 or Rdcvf2 mRNA is referred to hereinas “decreased transcription.”

A “host cell,” as used herein, refers to a prokaryotic or eukaryoticcell that contains heterologous DNA that has been introduced into thecell by any means, e.g., electroporation, calcium phosphateprecipitation, microinjection, transformation, viral infection, and thelike.

“Heterologous” as used herein means “of different natural origin” orrepresent a non-natural state. For example, if a host cell istransfotined with a DNA or gene derived from another organism,particularly from another species, that gene is heterologous withrespect to that host cell and also with respect to descendants of thehost cell which carry that gene. Similarly, heterologous refers to anucleotide sequence derived from and inserted into the same natural,original cell type, but which is present in a non-natural state, e.g. adifferent copy number, or under the control of different regulatoryelements.

A vector molecule is a nucleic acid molecule into which heterologousnucleic acid may be inserted which can then be introduced into anappropriate host cell. Vectors preferably have one or more origin ofreplication, and one or more site into which the recombinant DNA can beinserted. Vectors often have convenient means by which cells withvectors can be selected from those without, e.g., they encode drugresistance genes. Common vectors include plasmids, viral genomes, and(primarily in yeast and bacteria) “artificial chromosomes.”

“Plasmids” generally are designated herein by a lower case p precededand/or followed by capital letters and/or numbers, in accordance withstandard naming conventions that are familiar to those of skill in theart. Starting plasmids disclosed herein are either commerciallyavailable, publicly available on an unrestricted basis, or can beconstructed from available plasmids by routine application of wellknown, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated, even ifsubsequently reintroduced into the natural system. Such polynucleotidescould be part of a vector and/or such polynucleotides or polypeptidescould be part of a composition, and still be isolated in that suchvector or composition is not part of its natural environment.

As used herein, the term “transcriptional control sequence” refers toDNA sequences, such as initiator sequences, enhancer sequences, andpromoter sequences, which induce, repress, or otherwise control thetranscription of protein encoding nucleic acid sequences to which theyare operably linked.

As used herein, “Rdcvf1 transcriptional control sequences” or “Rdcvf2transcriptional control sequences” are any of those transcriptionalcontrol sequences normally found associated with a mammalian Rdcvf1 orRdcvf2 gene, preferably with the Rdcvf2 gene as found in the mouse orhuman genome.

As used herein, “non-human transcriptional control sequence” is anytranscriptional control sequence not found in the human genome.

The term “polypeptide” is used interchangeably herein with the terms“polypeptides” and “protein(s)”.

As used herein, a “chemical derivative” of a polypeptide of theinvention is a polypeptide of the invention that contains additionalchemical moieties not normally a part of the molecule. Such moieties mayimprove the molecule's solubility, absorption, biological half life,etc. The moieties may alternatively decrease the toxicity of themolecule, eliminate or attenuate any undesirable side effect of themolecule, etc. Moieties capable of mediating such effects are disclosed,for example, in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980).

As used herein, a “neuroprotective agent” is a compound that prevents orprotects neuronal cells from degeneration. A “retinoprotective agent” isa compound that prevents or protects retinal cells from degeneration.

The invention includes nucleic acid molecules, preferably DNA molecules,such as (1) an isolated comprising a nucleotide sequence as set forth inSEQ ID NO:1 or SEQ ID NO:3, (2) isolated nucleic acid molecules thatcomprise nucleic acid sequences that hybridize under high stringencyconditions to the isolated DNA as set forth in SEQ ID NO:1 or SEQ IDNO:3, and (3) nucleic acid sequences that hybridize to (1) or (2),above. Such hybridization conditions may be highly stringent or lesshighly stringent, as described above. In instances wherein the nucleicacid molecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).Suitable ranges of such stringency conditions for nucleic acids ofvarying compositions are described in Krause and Aaronson (1991) Methodsin Enzymology, 200:546-556 in addition to Maniatis et al., cited above.

These nucleic acid molecules may act as target gene antisense molecules,useful, for example, in target gene regulation and/or as antisenseprimers in amplification reactions of target gene nucleic acidsequences. Further, such sequences may be used as part of ribozymeand/or triple helix sequences, also useful for target gene regulation.Still further, such molecules may be used as components of diagnosticmethods whereby the presence of a RdCVF1 or RdCVF2 disease-causingallele, may be detected.

The invention also encompasses (a) vectors that contain any of theforegoing coding sequences (i.e., sense) and/or their complements (i.e.,antisense); (b) expression vectors that contain any of the foregoingcoding sequences operatively associated with a regulatory element thatdirects the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

The invention includes fragments of any of the nucleic acid sequencesdisclosed herein. Fragments of the full length Rdcvf1 or Rdcvf2 gene maybe used as a hybridization probe for a cDNA library to isolate the fulllength gene and to isolate other genes which have a high sequencesimilarity to the Rdcvf1 or Rdcvf2 gene and similar biological activity.Probes of this type preferably have at least about 30 bases and maycontain, for example, from about 30 to about 50 bases, about 50 to about100 bases, about 100 to about 200 bases, or more than 200 bases (e.g.300). The probe may also be used to identify a cDNA clone correspondingto a full length transcript and a genomic clone or clones that containthe complete Rdcvf1 or Rdcvf2 gene including regulatory and promoterregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the Rdcvf1 or Rdcvf2 gene by using the known DNAsequence to synthesize an oligonucleotide probe or random priming of theisolated sequence disclosed in FIGS. 1 to 8. Labeled oligonucleotideshaving a sequence complementary to that of the gene of the presentinvention are used to screen a library of human cDNA, genomic DNA todetermine which individual clones of the library the probe hybridizesto.

In addition to the gene sequences described above, orthologs of suchsequences, as may, for example be present in other species, may beidentified and may be readily isolated, without undue experimentation,by molecular biological techniques well known in the art. Further, theremay exist genes at other genetic loci within the genome that encodeproteins which have extensive homology (homologs) to one or more domainsof such gene products. These genes may also be identified via similartechniques. Examples of orthologs or homologues are provided in FIG. 8,10, 11, 12 or 13.

For example, the isolated expressed gene sequence may be labeled andused to screen a cDNA library constructed from mRNA obtained from theorganism of interest. Hybridization conditions will be of a lowerstringency when the cDNA library was derived from an organism differentfrom the type of organism from which the labeled sequence was derived.Alternatively, the labeled fragment may be used to screen a genomiclibrary derived from the organism of interest, again, usingappropriately low stringent conditions. Such low stringency conditionswill be well known to those of skill in the art, and will varypredictably depending on the phylogeny of specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al. cited above.

Further, a previously unknown expressed gene-type sequence may beisolated by performing PCR using two degenerated oligonucleotide primerpools designed on the basis of amino acid sequences within the gene ofinterest. The template for the reaction may be cDNA obtained by reversetranscription of mRNA prepared from human or nonhuman cell lines ortissue known or suspected to express a homologue or splicing variants.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of a expressed gene-likenucleic acid sequence. The PCR fragment may then be used to isolate afull length cDNA clone by a variety of methods. For example, theamplified fragment may be labeled and used to screen a bacteriophagecDNA library. Alternatively, the labeled fragment may be used to screena genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction may be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” with guanines using a standardterminal transferase reaction, the hybrid may be digested with RNAase H,and second strand synthesis may then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment may easily beisolated. For a review of cloning strategies which may be used, seee.g., Sambrook et al., 1989, supra.

In cases where the differentially expressed gene identified is thenormal, or wild type, gene, this gene may be used to isolate mutantalleles of the gene. Such an isolation is preferable in processes anddisorders which are known or suspected to have a genetic basis. Mutantalleles may be isolated from individuals either known or suspected tohave a genotype which contributes to disease symptoms. Mutant allelesand mutant allele products may then be utilized in the diagnostic assaysystems described below.

A cDNA of the mutant gene may be isolated, for example, by using RT-PCR,a technique which is well known to those of skill in the art. In thiscase, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide (or random hexamers) to mRNA isolated fromtissue known or suspected to be expressed in an individual putativelycarrying the mutant allele, and by extending the new strand with reversetranscriptase. The second strand of the cDNA is then synthesized usingan oligonucleotide that hybridizes specifically to the 5′ end of thenormal gene (or by any other means). Using these two primers, theproduct is then amplified via PCR, cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well known to thoseof skill in the art. By comparing the DNA sequence of the mutant gene tothat of the normal gene, the mutation(s) responsible for the loss oralteration of function of the mutant gene product can be ascertained.

Alternatively, a genomic or cDNA library can be constructed and screenedusing DNA or RNA, from a tissue known to or suspected of expressing thegene of interest in an individual suspected of to carry the mutantallele. The normal gene or any suitable fragment thereof may then belabeled and used as a probe to identify the corresponding mutant allelein the library. The clone containing this gene may then be purifiedthrough methods routinely practiced in the art, and subjected tosequence analysis as described above.

Additionally, an expression library can be constructed utilizing DNAisolated from or cDNA synthesized from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of to carrythe mutant allele. In this manner, gene products made by the putativelymutant tissue may be expressed and screened using standard antibodyscreening techniques in conjunction with antibodies raised against thenormal gene product, as described, below. (For screening techniques,see, for example, Harlow, E. and Lane, eds., 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Press, Cold Spring Harbor.) Incases where the mutation results in an expressed gene product withaltered function (e.g., as a result of a missense mutation), apolyclonal set of antibodies are likely to cross-react with the mutantgene product. Library clones detected via their reaction with suchlabeled antibodies can be purified and subjected to sequence analysis asdescribed above.

Differentially expressed gene products include those proteins encoded bynucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13, in particular, apolypeptide that is or includes the amino acid sequence set out in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12 or SEQ ID NO:14, or fragments thereof.

In addition, expressed gene products may include proteins that representfunctionally equivalent gene products. Such an equivalent gene productmay contain deletions, additions or substitutions of amino acid residueswithin the amino acid sequence encoded by the differentially expressedgene sequences described, above, but which result in a silent change,thus producing a functionally equivalent differentially expressed geneproduct (polymorphisms). Amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involvedand on comparison with amino-acids sequence from other species.

For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Functionally equivalent,” as utilized herein, may referto a protein or polypeptide capable of exhibiting a substantiallysimilar in vivo or in vitro activity as the endogenous differentiallyexpressed gene products encoded by the differentially expressed genesequences described above. “Functionally equivalent” may also refer toproteins or polypeptides capable of interacting with other cellular orextracellular molecules in a manner similar to the way in which thecorresponding portion of the endogenous differentially expressed geneproduct would. For example, a “functionally equivalent” peptide would beable, in an immunoassay, to diminish the binding of an antibody to thecorresponding peptide (i.e., the peptidic amino acid sequence of whichwas modified to achieve the “functionally equivalent” peptide) of theendogenous protein, or to the endogenous protein itself, where theantibody was raised against the corresponding peptide of the endogenousprotein. An equimolar concentration of the functionally equivalentpeptide will diminish the aforesaid binding of the corresponding peptideby at least about 5%, preferably between about 5% and 10%, morepreferably between about 10% and 25%, even more preferably between about25% and 50%, and most preferably between about 40% and 50%.

The differentially expressed gene products may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the differentially expressed gene polypeptides andpeptides of the invention by expressing nucleic acid encodingdifferentially expressed gene sequences are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing expressed gene protein codingsequences and appropriate transcriptional/translational control signals.These methods include, for example, in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Sambrook et al., 1989,supra, and Ausubel et al., 1989, supra. Alternatively, RNA or cDNAcapable of encoding expressed gene protein sequences may be chemicallysynthesized using, for example, synthesizers. See, for example, thetechniques described in “Oligonucleotide Synthesis”, 1984, Gait, M. J.ed., IRL Press, Oxford, which is incorporated by reference herein in itsentirety.

A variety of host-expression vector systems may be utilized to expressthe differentially expressed gene coding sequences of the invention.Such host-expression systems represent vehicles by which the codingsequences of interest may be produced and subsequently purified, butalso represent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express the differentiallyexpressed gene protein of the invention in situ. These include but arenot limited to microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing differentially expressed geneprotein coding sequences; yeast (e.g. Saccharomyces, Pichia) transformedwith recombinant yeast expression vectors containing the differentiallyexpressed gene protein coding sequences; insect cell systems infectedwith recombinant virus expression vectors (e.g., baculovirus) containingthe differentially expressed gene protein coding sequences; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid transformation vectors (e.g., Tiplasmid) containing differentially expressed gene protein codingsequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothioneine promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter; Cytomegalovirus Early gene promoter).

Expression of RDCVF1 or RDCVF2 by a cell from an Rdcvf1 or Rdcvf2 genethat is native to a the cell can also be performed. Methods for suchexpression are detailed in, e.g., U.S. Pat. Nos. 5,641,670; 5,733,761;5,968,502; and 5,994,127, all of which are expressly incorporated byreference herein in their entirety. Cells that have been induced toexpress RDCVF1 or RDCVF2 by the methods of any of U.S. Pat. Nos.5,641,670; 5,733,761; 5,968,502; and 5,994,127 can be implanted into adesired tissue in a living animal in order to increase the localconcentration of RDCVF1 or RDCVF2 in the tissue.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for thedifferentially expressed gene protein being expressed. For example, whena large quantity of such a protein is to be produced, for the generationof antibodies or to screen peptide libraries, for example, vectors whichdirect the expression of high levels of fusion protein products that arereadily purified may be desirable. Such vectors include, but are notlimited, to the E. coli expression vector pUR278 (Ruther et al., 1983,EMBO J. 2:1791), in which the differentially expressed gene proteincoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.PGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-asephagarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene protein can be released from the GST moiety by using theseendopeptidases.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase (“cat”) or luciferasetranscription unit, downstream of restriction site or sites forintroducing a candidate promoter fragment; i.e., a fragment that maycontain a promoter. As is well known, introduction into the vector of apromoter-containing fragment at the restriction site upstream of the cator luciferase gene engenders production of CAT or luciferase activity,which can be detected by standard CAT assays or luminometry. Vectorssuitable to this end are well known and readily available. Three suchvectors are pKK232-8, -pCM7 and pGL3 (Promega, E1751, Genebank Ass n°u47295). Thus, promoters for expression of polynucleotides of thepresent invention include not only well known and readily availablepromoters, but also promoters that readily may be obtained by theforegoing technique, using a reporter gene assay.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lad and lacZ promoters, the T3 and T7promoters, the T5 tac promoter, the lambda PR, PL promoters and the trppromoter. Among known eukaryotic promoters suitable in this regard arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), and metallothionein promoters,such as the mouse metallothionein-I promoter.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is one of several insect systems that can be used as a vector toexpress foreign genes. The virus grows in Spodoptera frugiperda cells.The differentially expressed gene coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of differentiallyexpressed gene coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed. (E.g., see Smith et al.,1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the differentially expressed gene coding sequence of interestmay be ligated to an adenovirus transcription/translation controlcomplex, e.g., the late promoter and tripartite leader sequence. Thischimeric gene may then be inserted in the adenovirus genome by in vitroor in vivo recombination. Insertion in a non-essential region of theviral genome (e.g., region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing differentially expressed geneprotein in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81:3655-3659). Specific initiation signals may also berequired for efficient translation of inserted differentially expressedgene coding sequences. These signals include the ATG initiation codonand adjacent sequences. In cases where an entire differentiallyexpressed gene, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of the differentially expressed gene codingsequence is inserted, exogenous translational control signals,including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals (Kozack sequence)and initiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987, Methods in Enzymol.153:516-544).

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forexpression vector construction, introduction of the vector into the hostand expression in the host per se are routine skills in the art.

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct may be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe differentially expressed protein may be engineered. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with DNA controlled by appropriateexpression control elements (e.g., promoter, enhancer, sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines. This method may advantageously be used to engineer cell lineswhich express the differentially expressed protein. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that affect the endogenous activity of the differentiallyexpressed protein. These stable cell lines might by used as a way ofcellular therapy directly or after encapsulation.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes.

An alternative fusion protein system allows for the ready purificationof non-denatured fusion proteins expressed in human cell lines(Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976). Inthis system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the gene's open reading frame istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

When used as a component in assay systems such as those described below,the differentially expressed protein may be labeled, either directly orindirectly, to facilitate detection of a complex formed between thedifferentially expressed protein and a test substance. Any of a varietyof suitable labeling systems may be used including but not limited toradioisotopes such as ¹²⁵I; enzyme labeling systems that generate adetectable calorimetric signal or light when exposed to substrate; andfluorescent labels.

Where recombinant DNA technology is used to produce the differentiallyexpressed protein for such assay systems, it may be advantageous toengineer fusion proteins that can facilitate labeling, immobilizationand/or detection.

Indirect labeling involves the use of a protein, such as a labeledantibody, which specifically binds to either a differentially expressedgene product. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments and fragments producedby an Fab expression library.

In another embodiment, nucleic acids comprising a sequence encodingRDCVF1 or RDCVF2 protein or functional derivative thereof, areadministered to promote cone function, by way of gene therapy. Genetherapy refers to therapy performed by the administration of a nucleicacid to a subject. In this embodiment of the invention, the nucleic acidproduces its encoded protein that mediates a therapeutic effect bypromoting cone function.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

In a preferred aspect, the therapeutic comprises a Rdcvf1 or Rdcvf2nucleic acid that is part of an expression vector that expresses aRDCVF1 or RDCVF2 protein or fragment or chimeric protein thereof in asuitable host. In particular, such a nucleic acid has a promoteroperably linked to the Rdcvf1 or Rdcvf2 coding region, said promoterbeing inducible or constitutive, and, optionally, tissue-specific. Inanother particular embodiment, a nucleic acid molecule is used in whichthe Rdcvf1 or Rdcvf2 coding sequences and any other desired sequencesare flanked by regions that promote homologous recombination at adesired site in the genome, thus providing for intrachromosomalexpression of the Rdcvf1 or Rdcvf2 nucleic acid (Koller and Smithies,1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989,Nature 342:435-438).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (adenovirus, adeno-associated virus and lentivirus) (see, e.g.,U.S. Pat. No. 4,980,286 and others mentioned infra), or by directinjection of naked DNA, or by use of microparticle bombardment (e.g., agene gun; Biolistic, Dupont), or coating with lipids or cell-surfacereceptors or transfecting agents, encapsulation in liposomes,microparticles, or microcapsules, or by administering it in linkage to apeptide which is known to enter the nucleus, by administering it inlinkage to a ligand subject to receptor-mediated endocytosis (see e.g.,U.S. Pat. Nos. 5,166,320; 5,728,399; 5,874,297; and 6,030,954, all ofwhich are incorporated by reference herein in their entirety) (which canbe used to target cell types specifically expressing the receptors),etc. In another embodiment, a nucleic acid-ligand complex can be formedin which the ligand comprises a fusogenic viral peptide to disruptendosomes, allowing the nucleic acid to avoid lysosomal degradation. Inyet another embodiment, the nucleic acid can be targeted in vivo forcell specific uptake and expression, by targeting a specific receptor(see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316;WO93/14188; and WO 93/20221). Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (see, e.g., U.S. Pat. Nos.5,413,923; 5,416,260; and 5,574,205; and Zijlstra et al., 1989, Nature342:435-438).

In a specific embodiment, a viral vector that contains the Rdcvf1 orRdcvf2 nucleic acid is used. For example, a retroviral vector can beused (see, e.g., U.S. Pat. Nos. 5,219,740; 5,604,090; and 5,834,182).These retroviral vectors have been modified to delete retroviralsequences that are not necessary for packaging of the viral genome andintegration into host cell DNA. The Rdcvf1 or Rdcvf2 nucleic acid to beused in gene therapy is cloned into the vector, which facilitatesdelivery of the gene into a patient.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Methods for conductingadenovirus-based gene therapy are described in, e.g., U.S. Pat. Nos.5,824,544; 5,868,040; 5,871,722; 5,880,102; 5,882,877; 5,885,808;5,932,210; 5,981,225; 5,994,106; 5,994,132; 5,994,134; 6,001,557; and6,033,8843, all of which are incorporated by reference herein in theirentirety.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy. Adeno-associated viruses are especially attractive vehicles fordelivering genes to the retina. Methods for producing and utilizing AAVare described, e.g., in U.S. Pat. Nos. 5,173,414; 5,252,479; 5,552,311;5,658,785; 5,763,416; 5,773,289; 5,843,742; 5,869,040; 5,942,496; and5,948,675, all of which are incorporated by reference herein in theirentirety.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient directly or after encapsulation.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells and may be used in accordance with the presentinvention, provided that the necessary developmental and physiologicalfunctions of the recipient cells are not disrupted. The technique shouldprovide for the stable transfer of the nucleic acid to the cell, so thatthe nucleic acid is expressible by the cell and preferably heritable andexpressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy, aRdcvf1 or Rdcvf2 nucleic acid is introduced into the cells such that itis expressible by the cells or their progeny, and the recombinant cellsare then administered in vivo for therapeutic effect. In a specificembodiment, stem or progenitor cells are used. Any stem- and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention. Such stem cells include but are not limited to hematopoieticstem cells (HSC), stem cells of epithelial tissues such as the skin andthe lining of the gut, embryonic heart muscle cells, liver stem cells(see, e.g., WO 94/08598), and neural stem cells (Stemple and Anderson,1992, Cell 71:973-985).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, 1980, Meth. Cell Bio. 21A:229). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Pittelkow andScott, 1986, Mayo Clinic Proc. 61:771). If the ESCs are provided by adonor, a method for suppression of host versus graft reactivity (e.g.,irradiation, drug or antibody administration to promote moderateimmunosuppression) can also be used. Retinal stem cells (Tropepe et al.,2000, Science, 287: 2032).

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment of the invention. Teclmiques by whichthis may be accomplished include (a) the isolation and establishment ofHSC cultures from bone marrow cells isolated from the future host, or adonor, or (b) the use of previously established long-term HSC cultures,which may be allogeneic or xenogeneic. Non-autologous HSC are usedpreferably in conjunction with a method of suppressing transplantationimmune reactions of the future host/patient. In a particular embodimentof the present invention, human bone marrow cells can be obtained fromthe posterior iliac crest by needle aspiration (see, e.g., Kodo et al.,1984, J. Clin. Invest. 73:1377-1384). In a preferred embodiment of thepresent invention, the HSCs can be made highly enriched or insubstantially pure form. This enrichment can be accomplished before,during, or after long-term culturing, and can be done by any techniquesknown in the art. Long-term cultures of bone marrow cells can beestablished and maintained by using, for example, modified Dexter cellculture techniques (Dexter et al., 1977, J. Cell Physiol. 91:335) orWitlock-Witte culture techniques (Witlock and Witte, 1982, Proc. Natl.Acad. Sci. USA 79:3608-3612).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Described herein are methods for the production of antibodies capable ofspecifically recognizing one or more differentially expressed geneepitopes. Such antibodies may include, but are not limited to polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection of a fingerprint,target, gene in a biological sample, or, alternatively, as a method forthe inhibition of abnormal target gene activity. Thus, such antibodiesmay be utilized as part of disease treatment methods, and/or may be usedas part of diagnostic techniques whereby patients may be tested forabnormal levels of Rdcvf1 or Rdcvf2, or for the presence of abnormalforms of Rdcvf1 or Rdcvf2 by sampling the aqueous humor and/or vitreousby methods familiar to those of skill in the art (e.g. Forster, R K,Abbott, R L, Gelender, H. (1980) Management of infectiousendophthalmitis Ophthalmology 87, 313-319)].

For the production of antibodies to a differentially expressed gene,various host animals may be immunized by injection with a differentiallyexpressed protein, or a portion thereof or by DNA immunization. Suchhost animals may include but are not limited to rabbits, mice, and rats,to name but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection withdifferentially expressed gene product supplemented with adjuvants asalso described above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableor hypervariable region derived from a murine mAb and a humanimmunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to producedifferentially expressed gene-single chain antibodies. Single chainantibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chainpolypeptide.

Most preferably, techniques useful for the production of “humanizedantibodies” can be adapted to produce antibodies to the polypeptides,fragments, derivatives, and functional equivalents disclosed herein.Such techniques are disclosed in U.S. Pat. Nos. 5,932,448; 5,693,762;5,693,761; 5,585,089; 5,530,101; 5,910,771; 5,569,825; 5,625,126;5,633,425; 5,789,650; 5,545,580; 5,661,016; and 5,770,429, thedisclosures of all of which are incorporated by reference herein intheir entirety.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Particularly preferred, for ease of detection, is the sandwich assay, ofwhich a number of variations exist, all of which are intended to beencompassed by the present invention.

For example, in a typical forward assay, unlabeled antibody isimmobilized on a solid substrate and the sample to be tested broughtinto contact with the bound molecule. After a suitable period ofincubation, for a period of time sufficient to allow formation of anantibody-antigen binary complex. At this point, a second antibody,labeled with a reporter molecule capable of inducing a detectablesignal, is then added and incubated, allowing time sufficient for theformation of a ternary complex of antibody-antigen-labeled antibody. Anyunreacted material is washed away, and the presence of the antigen isdetermined by observation of a signal, or may be quantitated bycomparing with a control sample containing known amounts of antigen.Variations on the forward assay include the simultaneous assay, in whichboth sample and antibody are added simultaneously to the bound antibody,or a reverse assay in which the labeled antibody and sample to be testedare first combined, incubated and added to the unlabeled surface boundantibody. These techniques are well known to those skilled in the art,and the possibility of minor variations will be readily apparent. Asused herein, “sandwich assay” is intended to encompass all variations onthe basic two-site technique. For the immunoassays of the presentinvention, the only limiting factor is that the unlabeled and thelabeled antibodies be an RdCVF1- or RdCVF2-specific antibody.

The most commonly used reporter molecules in this type of assay areeither enzymes, fluorophore- or radionuclide-containing molecules. Inthe case of an enzyme immunoassay an enzyme is conjugated to the secondantibody, usually by means of glutaraldehyde or periodate. As will bereadily recognized, however, a wide variety of different ligationtechniques exist, which are well-known to the skilled artisan. Commonlyused enzymes include horseradish peroxidase, glucose oxidase,beta-galactosidase and alkaline phosphatase, among others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase conjugates; for peroxidaseconjugates, 1,2-phenylenediamine or toluidine are commonly used. It isalso possible to employ fluorogenic substrates, which yield afluorescent product rather than the chromogenic substrates noted above.A solution containing the appropriate substrate is then added to thetertiary complex of antibody-RdCVF1- or RdCVF2-labeled antibody. Thesubstrate reacts with the enzyme linked to the second antibody, giving aqualitative visual signal, which may be further quantitated, usuallyspectrophotometrically, to give an evaluation of the amount of Rdcvf1 orRdcvf2 which is present in the serum sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.Immunofluorescence and EIA techniques are both very well established inthe art and are particularly preferred for the present method. However,other reporter molecules, such as radioisotopes, chemiluminescent orbioluminescent molecules may also be employed. It will be readilyapparent to the skilled artisan how to vary the procedure to suit therequired use.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of thegene encoding a polypeptide selected from the group consisting of thepolypeptides set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:14 which is associatedwith a dysfunction will provide a diagnostic tool that can add to, ordefine, a diagnosis of a disease, or susceptibility to a disease, whichresults from underexpression, over-expression or altered spatial ortemporal expression of the gene. Individuals carrying mutations in thegene may be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled nucleotide sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in electrophoreticmobility of DNA fragments in gels, with or without denaturing agents(SSCP), or by direct DNA sequencing (e.g., Myers et al., Science (1985)230:1242). Sequence changes at specific locations may also be revealedby nuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA(1985) 85: 4397-4401). In another embodiment, an array ofoligonucleotides probes comprising the Rdcvf1 or Rdcvf2 nucleotidesequence or fragments thereof can be constructed to conduct efficientscreening of e.g., genetic mutations. Array technology methods are wellknown and have general applicability and can be used to address avariety of questions in molecular genetics including gene expression,genetic linkage, and genetic variability (see for example: M. Chee etal., Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to disease through detection of mutation in the Rdcvf1 orRdcvf2 gene by the methods described. In addition, such diseases may bediagnosed by methods comprising determining from a sample derived from asubject an abnormal Rdcvf1 or Rdcvf2 expression: Expression can bemeasured at the RNA level using any of the methods well known in the artfor the quantitation of polynucleotides, such as, for example, nucleicacid amplification, for instance PCR, RT-PCR, RNase protection, Northernblotting and other hybridization methods. Assay techniques that can beused to determine levels of a protein, such as a polypeptide of thepresent invention, in a sample derived from a host are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostickit which comprises:

-   -   (a) a polynucleotide of the present invention, preferably the        nucleotide sequence encoding a polypeptide selected from the        group consisting of the polypeptides set forth in SEQ ID NO:2,        SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID        NO:12 and SEQ ID NO:14, or a fragment thereof;    -   (b) a nucleotide sequence complementary to that of (a);    -   (c) a polypeptide of the present invention, preferably the        polypeptide of or a fragment thereof; or    -   (d) an antibody to a polypeptide of the present invention,        preferably to a polypeptide selected from the group consisting        of the polypeptides set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ        ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID        NO:14.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularlyretinitis pigmentosa, age-related macular degeneration, Bardet-Biedelsyndrome, Bassen-kornzweig syndrome, best disease, choroidema, gyrateatrophy, congenital amourosis, refsun syndrome, stargardt disease andUsher syndrome. The nucleotide sequences of the present invention arealso valuable for chromosome localization. The chromosomal location canbe obtained by PCR on DNA prepared from a panel of hybrid cell lines(mouse-hamster). The chromosomal location of the human gene can bepredicted from the location of the mouse gene by syntheny (McCarthy etal. (1997), Genome research, 7, 1153). The sequence is specificallytargeted to, and can hybridize with, a particular location on anindividual chromosome, including a mouse or a human chromosome. Themapping of relevant sequences to chromosomes according to the presentinvention is an important first step in correlating those sequences withgene associated disease. Once a sequence has been mapped to a precisechromosomal location, the physical position of the sequence on thechromosome can be correlated with genetic map data. Such data are foundin, for example, V. McKusick, Mendelian Inheritance in Man (availableon-line through Johns Hopkins University Welch Medical Library, RetNet).The relationship between genes and diseases that have been mapped to thesame chromosomal region are then identified through linkage analysis(coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of Rdcvf1 or Rdcvf2,mimetics or agonists. The compositions may be administered alone or incombination with at least one other agent, such as stabilizing compound,which may be administered in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. The compositions may be administered to a patientalone, or in combination with other agents, drugs or hormones.

The pharmaceutical compositions encompassed by the invention may beadministered by any number of routes: e.g. Transscleral delivery ofbioreactive protein to the choroid and retina Ambati et al. (2000)Investigative Ophthalmology and Visual Science, 41, 1186. The fotmulation of topical ophthalmic preparations, including ophthalmicsolutions, suspensions and ointments is well known to those skilled inthe art (see Remington's Pharmaceutical Sciences, 18th Edition, Chapter86, pages 1581-1592 Mack Publishing Company, 1990). Other modes ofadministration are available, including intracameral injections (whichmay be made directly into the anterior chamber or directly into thevitreous chamber), subconjunctival injections and retrobulbarinjections, and methods and means for producing ophthalmic preparationssuitable for such modes of administration are also well known.

As used in this application, “extraocular” refers to the ocular surfaceand the (external) space between the eyeball and the eyelid. Examples ofextraocular regions include the eyelid formix or cul-de-sac, theconjunctival surface and the corneal surface. This location is externalto all ocular tissue and an invasive procedure is not required to accessthis region. Examples of extraocular systems include inserts and“topically” applied drops, gels or ointments which may be used todeliver therapeutic material to these regions. Extraocular devices aregenerally easily removable, even by the patient.

The following patents disclose extraocular systems which are used toadminister drugs to the extraocular regions. Higuchi et al. discloses inU.S. Pat. Nos. 3,981,303, 3,986,510 and 3,995,635, a biodegradableocular insert which contains a drug. The insert can be made in differentshapes for retention in the cul-de-sac of the eyeball, the extraocularspace between the eyeball and the eyelid. Several common biocompatiblepolymers are disclosed as suitable for use in fabricating this device.These polymers include zinc alginate, poly (lactic acid), poly (vinylalcohol), poly (anhydrides) and poly (glycolic acid). The patents alsodescribe membrane coated devices with reduced permeation to the drug andhollow chambers holding the drug formulation.

Theeuwes, U.S. Pat. No. 4,217,898, discloses microporous reservoirswhich are used for controlled drug delivery. These devices are placedextraocularly in the ocular cul-de-sac. Among the polymer systems ofinterest include poly (vinylchloride)-co-poly (vinyl acetate)copolymers. Kaufman discloses in U.S. Pat. Nos. 4,865,846 and 4,882,150an ophthalmic drug delivery system which contains at least onebio-erodible material or ointment carrier for the conjunctival sac. Thepatent discloses polymer systems, such as, poly (lactide), poly(glycolide), poly (vinyl alcohol) and cross linked collagen, as suitabledelivery systems.

In the presently described use of RDCVF1 or RDCVF2 protein product forthe treatment of retinal disease or injury it is also advantageous thata topically applied ophthalmic formulation include an agent to promotethe penetration or transport of the therapeutic agent into the eye. Suchagents are known in the art. For example, Ke et al., U.S. Pat. No.5,221,696 disclose the use of materials to enhance the penetration ofophthalmic preparations through the cornea.

Intraocular systems are those systems which are suitable for use in anytissue compartment within, between or around the tissue layers of theeye itself. These locations include subconjunctival (under the ocularmucous membrane adjacent to the eyeball), orbital (behind the eyeball),and intracameral (within the chambers of the eyeball itself). Incontrast to extraocular systems, an invasive procedure consisting ofinjection or implantation is required to access these regions.

The following patents disclose intraocular devices. Wong, U.S. Pat. No.4,853,224, discloses microencapsulated drugs for introduction into thechamber of the eye. Polymers which are used in this system includepolyesters and polyethers. Lee, U.S. Pat. No. 4,863,457, discloses abiodegradable device which is surgically implanted intraocularly for thesustained release of therapeutic agents. The device is designed forsurgical implantation under the conjunctiva (mucous membrane of theeyeball). Krezancaki, U.S. Pat. No. 4,188,373, discloses apharmaceutical vehicle which gels at human body temperature. Thisvehicle is an aqueous suspension of the drug and gums or cellulosederived synthetic derivatives. Haslam et al. discloses in U.S. Pat. Nos.4,474,751 and 4,474,752 a polymer-drug system which is liquid at roomtemperature and gels at body temperature. Suitable polymers used in thissystem include polyoxyethylene and polyoxy propylene. Davis et al.disclose in U.S. Pat. No. 5,384,333 a biodegradable injectable drugdelivery polymer which provides long term drug release. The drugcomposition is made up of a pharmaceutically active agent in abiodegradable polymer matrix, where the polymer matrix is a solid attemperatures in the range 20.degree. to 37.degree. C. and is flowable attemperatures in the range 38.degree. to 52.degree. C. The drug deliverypolymer is not limited to the delivery of soluble or liquid drugformulations. For example, the polymer can be used as a matrix forstabilizing and retaining at the site of injection drug-containingmicrospheres, liposomes or other particulate-bound drugs.

A particularly suitable vehicle for intraocular injection is steriledistilled water in which the RDCVF1 or RDCVF2 protein product isformulated as a sterile, isotonic solution, properly preserved. Yetanother ophthalmic preparation may involve the formulation of the RDCVF1or RDCVF2 protein product with an agent, such as injectable microspheresor liposomes, that provides for the slow or sustained release of theprotein which may then be delivered as a depot injection. Other suitablemeans for the intraocular introduction of RDCVF1 or RDCVF2 proteinproduct includes, implantable drug delivery devices or which contain theRDCVF1 or RDCVF2 protein product.

The ophthalmic preparations of the present invention, particularlytopical preparations, may include other components, for exampleophthalmically acceptable preservatives, tonicity agents, cosolvents,wetting agents, complexing agents, buffering agents, antimicrobials,antioxidants and surfactants, as are well known in the art. For example,suitable tonicity enhancing agents include alkali metal halides(preferably sodium or potassium chloride), mannitol, sorbitol and thelike. Sufficient tonicity enhancing agent is advantageously added sothat the formulation to be instilled into the eye is hypotonic orsubstantially isotonic. Suitable preservatives include, but are notlimited to, benzalkonium chloride, thimerosal, phenethyl alcohol,methylparaben, propylparaben, chlorhexidine, sorbic acid and the like.Hydrogen peroxide may also be used as preservative. Suitable cosolventsinclude, but are not limited to, glycerin, propylene glycol andpolyethylene glycol. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude, but are not limited to, sorbitan esters, polysorbates such aspolysorbate 80, tromethamine, lecithin, cholesterol, tyloxapol and thelike. The buffers can be conventional buffers such as borate, citrate,phosphate, bicarbonate, or Tris-HCl.

The formulation components are present in concentrations that areacceptable to the extraocular or intraocular site of administration. Forexample, buffers are used to maintain the composition at physiologicalpH or at slightly lower pH, typically within a pH range of from about 5to about 8. Additional formulation components may include materialswhich provide for the prolonged ocular residence of the extraocularlyadministered therapeutic agent so as to maximize the topical contact andpromote absorbtion. Suitable materials include polymers or gel formingmaterials which provide for increased viscosity of the ophthalmicpreparation. Chitosan is a particularly suitable material as an ocularrelease-rate controlling agent in sustained release liquid ophthalmicdrug formulations (see U.S. Pat. No. 5,422,116, Yen, et. al.) Thesuitability of the formulations of the instant invention for controlledrelease (e.g., sustained and prolonged delivery) of an ophthalmictreating agent in the eye can be determined by various procedures knownin the art, e.g., as described in Journal of Controlled Release,6:367-373, 1987, as well as variations thereof.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated m aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a maimer that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of Rdcvf1 or Rdcvf2, such labeling wouldinclude amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example Rdcvf1 or Rdcvf2 or fragments thereof,antibodies to Rdcvf1, agonists, which ameliorates the symptoms orcondition. Therapeutic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation) and LD50 (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD50/ED50. Pharmaceuticalcompositions which exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage contained insuch compositions is preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage form employed,sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc. Pharmaceutical formulations suitable fororal administration of proteins are described, e.g., in U.S. Pat. Nos.5,008,114; 5,505,962; 5,641,515; 5,681,811; 5,700,486; 5,766,633;5,792,451; 5,853,748; 5,972,387; 5,976,569; and 6,051,561.

The following Examples illustrate the present invention, without in anyway limiting the scope thereof.

EXAMPLES 1. Expression Cloning

1) Total ARN Purification from Five Weeks Normal Mouse Retinas:

The cDNA library was constructed from retinas of Five weeks oldC57BL/6@N mice generally according to the method of Glissin et al(1974), Biochemistry, 13, 2633-2637. Briefly, after killing, animalswere enucleated and the eyes were first placed in Phosphate BufferSaline (PBS) supplemented with 0.1% DiEthyl PyroCarbonate (DEPC). Neuralretina was quickly dissected (the retinal pigmented epithelium wasomitted in this tissue preparation). Quickly after each dissection,tissues were homogenised in fresh 6 M Guanidinium Chloride. Ten retinaswere pooled in 2.4 ml GC in 4 ml sterile tubes, and the tissue wasdisrupted completely by strong homogenisation for 1 minute at roomtemperature.

Messenger RNA (mRNA) Purification from Five Weeks Normal Mouse Retina:

mRNA was isolated on oligo-dT coated porous beads (Oligotex, Qiagen)under stringent conditions according to the method of Kuribayashi et al,(1988). Nucleic Acids Res. Symposium series, 19, 61-64. Briefly, 100-150μg total mouse retina RNA were mixed with 15 μl of oligo-dT beads inBinding Buffer [10 mM Tris pH 7.5; 0.3 M NaCl; 0.1 M EDTA; 0.5% w/vSodium Duodecyl Sulfate (SDS)] and incubated 6 minutes at 65° C. in 0.5l becher of water, then progressively cooled to room temperature forabout 3-4 hours, then centrifuged at room temperature to recover theagarose beads. They were then washed twice by incubating 10 minutes in0.4 ml of (0.1 M Tris pH 7.5; 0.1 M NaCl; 1 mM EDTA; 0.5% w/v SDS).Bounded RNA (mRNA) were eluted in two steps with 50 μl of 70° C.-warmedRNAse-free water, precipitated with 10 μl Sodium Acetate pH 5.2 and 0.25ml ethanol and incubated 12 hours at −70° C. mRNA were collected bycentrifugation (1 hour at 15.000 rpm, followed by two washes with 70%ethanol) and resuspended in 20 μl of RNAse-free water. mRNAconcentration was measured at 260 nm and the absence of rRNAcontaminations was checked by gel electrophoresis under denaturatingcondition as above.

cDNA Synthesis:

was carried out according to the method of Okayama and Berg, (1982), MolCell Biol., 2, 161-170. First strand synthesis was primed with 2.5 μg ofNotI adaptor oligonucleotide (5′TGTTACCAATCTGAAGTGGGAGCGGCCGACAA(T)₁₈ 3′(SEQ ID NO: 28)) and incubated for 2 hours with 50 units of modifiedMoloney Murine Leukaemia virus (M-MLV) reverse transcriptase(Superscript II, Life Technology) under the conditions recommended bythe supplier. For second strand synthesis, reaction was incubated 4hours at 14° C. with 4 units of RNAseH and 100 units of DNA polymerase Iin SS Buffer [40 mM Tris pH 7.2; 85 mM Potassium Chloride; 4.4 mMMagnesium chloride; 3 mM DTT; 5 μg/ml Bovin Serum Albumin (BSA)] in afinal volume of 0.25 ml. EcoRI adapters (5′-OHAATTCGGCACGAGG 3′-OH (SEQID NO: 29)/3′-OH GCCGTGCTCC5′-PO₄ (SEQ ID NO: 30)) were ligated at bothends of double strand cDNA in 14 hours at 16° C. using 40 units of T4DNA ligase (Promega, Madison, USA) in a total volume of 20 μl under theconditions recommended by the supplier. The products of this reactionare dscDNA that have an EcoRI half site in 5′ and NotI half site in 3′that can be oriented in the cloning vector.

Ligation of dscDNA in pcDNA3:

was performed according to Maniatis T. (1992), Molecular cloning: alaboratory manual, 2nd ed., using 10 μg of pcDNA3 plasmid (Invitrogen)prepared by cutting with EcoRI and NotI (Promega, Madison, USA) underthe conditions specified by the supplier.

Propagation of Recombinant Clones:

was performed generally according to the method of Birnboim et al(1979), Nucleic Acids Res., 7, 1513-1523. Briefly, In order to makepools of 100 primary clones, we slightly modified the XL1 Gold(Strategene) transformation protocol (provided by Stratagene) asfollowed. After incubation in growth medium, the transformation reactionwas brought to 20% (vol./vol.) glycerol and 8% (vol./vol.) horse serumalbumin (HAS, Life Technologies). HAS and glycerol prevent deathfollowed freezing thawing. A titration was done by platting on agarplates (100 μg/ml ampicillin) increasing volume of each transformationreaction to calculate the volume giving 100 colonies, while the bulktransformation reaction was stored at −80° C. Recombinant plasmids fromthe library were purified by 96 at once. In order to prepare 96 pools of100 clones, the calculated volume corresponding to 100 clones wasplatted on agar and grown 20 hours at 37° C. The DNA was purifieddirectly from colonies striped from agar plates. A stock of each cultureat 23% glycerol was stored at −80° C. The DNA was purified using Qiawellultra (Qiagen) using a protocol recommended by the supplier. Typically,10 μg of purified plasmid were obtained, the concentration of eachpreparation was measured using optical density at 260 nm. To sub-divideselected pools of 100 into pools of 10, 50 μl of a 1/250.000 dilution ofthe glycerol stock from the original pool was plate on an agar plate at100 μg/ml ampicillin. After growth 16 hours at 37° C., individual 160colonies were replicated on 16 agar plates (10 per plate) and grown 16hours at 37° C. The 10 colonies from each plate were harvested and grownin liquid medium [Luria Broth (LB), 100 ug/ml ampicillin] for 3 hours at37° C. A stock of these cultures at 30% glycerol was stored at −80° C.Plasmid DNA was prepared as before. To divide sub-pools of 10 intoindividual clones, 50 μl of a 1/250.000 dilution of the glycerol stockof the sub-pools of 10 was plated onto agar plate with 100 μg/mlampicillin. After growth 16 hours at 37° C., 16 individual colonies werepicked and grown 16 hours in 2 ml LB 100 μg/ml ampicillin. A stock ofthese cultures at 30% glycerol was stored at −80° C. Plasmid DNA wasprepared as before.

Transient Transfection in Cos-1 Cells:

was performed using the method of Chen and Okayama, (1987).High-efficiency transformation of mammalian cells by plasmid DNA (MolCell Biol., 7, 2745-2752).

Chick Embryo Retinal Cultures:

The protocol was adapted from Adler and Hatlee [(1989) Science, 243,391]. Chick embryonic retina (6 days in ovo) is dissociated and platedin monolayer culture. Under these culture conditions with the absence ofdifferentiation signals, cones represent 60-80% of cells. We haveproduced polyclonal antibodies into rabbit against visinin (a chickencone marker, Genbank accession number M84729) and verified that theproportion of cones in our culture is of 60-80%. The simple environmentof our model (chemically defined medium, absence of cell to cellcontacts) in addition to the ease and speed of the method make it a veryappropriate system to study trophic factors involved in cone survival.Briefly, Retinas from embryos issued from a control isolate ofprogenitors, are dissected after six days of development in ovo, cellsdissociated and plated at low density (10⁵ cells/cm²). During ten dayscell viability (60-80% cones) was followed using LIVE/DEAD assay(Molecular probes, Eugene, USA) an assay that quantifies live and deadcells. The number of cells alive decreases down to 8% of initial cellsnumber after seven days in culture in chemically defined medium. Whenperformed in the presence of conditioned media from COS1 cellstransfected with pools of clones from the library, live cells arecounted after seven days in vitro.Chicken progenitors (strain 657 red label) were maintained in aseparated compartment for the purpose of this experiment in a hatchingfacility 25 km from the laboratory. Fertilized eggs obtained naturallywere collected weekly, and maintained at 17° C. (their biological zero)in the laboratory after hatching. Daily, 5 eggs are incubated for 24hours at 20° C. then 136 hours at 37° C. with intermittent reversion ofthe inclination of the eggs in a humidified chamber. The day of theculture, eggs surface are washed with Mucocit-A then broken, the chickenembryos are transferred in PBS. The stage of development of the eachembryo is verified be the 29^(th) by visual comparison to Hamburger andHamilton (1951), in (Essential Development Biology, Stem and HollandEd.). Two of the embryos were chosen and enucleated, the eyestransferred in CO₂-independent medium (Life technologies). Retinas weredissected and transferred in Ringer buffer and washed twice. Retinas arecut in small pieces and treated 20 minutes at 37° C. with a solution oftrypsin (0.25% w/v). The reaction is stopped by addition of culturemedia (M199, Life Technologies) supplemented with 10% inactivated FCS.The cell suspension is treated for few minutes in 25 μl of DNAse I (1mg/ml, Sigma). The cell suspension is then washed twice in ChemicalDefined Culture Media [CDCM, equal volumes of DMEM and M199 media (LifeTechnologies) and AB with Supplements (5 μg/ml Insulin; 5 μg/mlTransferring; 64 nM Progesterone; 0.1 mM Putrescin; 5 ng/ml Selenium; 3mM Taurine; 2.7 μM Cytidine 5′-diphosphoethanolamine; 5.2 μM Cytidine5′-diphosphocholine, 0.2 μg/ml Hydrocortisone; 30 nM3,3′-5-triiodo-L-thyronine; 1 mM Sodium Pyruvate), 0.3 μM ProstaglandinD₂; 0.1 mg/ml linoleic acid] in order to remove the FCS. Theconcentration of cells stained with trypan blue is measured withMallassez'cell and brought to two concentrations (5.6 and 1.12 10⁵cells/ml) corresponding to the two plating densities (2 and 4 10⁵cells/cm²).Conditioned media from Cos-1 transfected cells are thaw on ice and 50 μltransferred to two 96 wells tissue culture treated black plate (CorningCostar) that have been coated a solution of 100 μg/ml Poly-L-lysine(Sigma) according the plan:First Round of Screening:

1 1 1 1 2 C 2 2 2 3 3 P C 3 3 4 4 4 C 4 5 5 5 5 6 C 6 6 6 7 7 C 7 7 8 88 8 C 9 9 9 9 10 C 10 10 10 11 11 11 C 11 12 12 12 12 C 13 13 13 13 1414 C 14 14 15 15 15 C 15 16 16 16 16 17 C 17 17 17 18 18 C 18 18 19 1919 19 P 20 20 20 C 20Where numbers refer to n° of pools of 100 clones, C to conditioned mediafrom Cos-1 cells transfected with the empty vector (pcDNA3) and P apositive control (conditioned media transfected with pcDNA-mouseGDNF.Second and Third Round of Screening:

x.(y).01 x.(y).01 x.(y).01 x.(y).01 x.(y).02 C x.(y).02 x.(y).02x.(y).02 x.(y).03 x.(y).03 P C x.(y).03 x.(y).03 x.(y).04 x.(y).04x.(y).04 C x.(y).04 x.(y).05 x.(y).05 x.(y).05 x.(y).05 x.(y).06 Cx.(y).06 x.(y).06 x.(y).06 x.(y).07 x.(y).07 C x.(y).07 x.(y).07x.(y).08 x.(y).08 x.(y).08 x.(y).08 C x.(y).09 x.(y).09 x.(y).09x.(y).09 x.(y).10 C x.(y).10 x.(y).10 x.(y).10 x.(y).11 x.(y).11x.(y).11 C x.(y).11 x.(y).12 x.(y).12 x.(y).12 x.(y).12 C x.(y).13x.(y).13 x.(y).13 x.(y).13 x.(y).14 x.(y).14 C x.(y).14 x.(y).14x.(y).15 x.(y).15 x.(y).15 C x.(y).15 x.(y).16 x.(y).16 x.(y).16x.(y).16 x.(y) C x.(y) x.(y) x.(y) C57 C57 C C57 C57 C3H C3H C3H C3H P 00 0 C 0Where x (second round) and y (third round) represent the n° of the poolsselected in the first and second round respectively. 01 to 16 representthe sub-pools used. x(y) represents the parental pool from which the 16pools derived. C is as is first round. P was modified as pCMVScript-CNTFin the second round and progressively to pcDNA-939.09.08 in the thirdround. 0 represents chick cone cells in CDCM media only. C57 and C3H,conditioned media from explants of retinas prepared as described in(preparing conditioned media from explants of mouse retinas) fromC57BL/6@N and C3H/He@N mouse retinas aged of 5 weeks respectively.50 μl of cell suspensions corresponding to two densities (2 and 4 10⁵cells/cm²) were added to the well of the two 96 wells plates filledconditioned media with a 8 channel motorized pipette (Biohit) in orderto minimized experimental errors. Cells were incubated for 7 days at 37°C. in 5% CO₂.

Preparing Conditioned Media from Explants of Mouse Retinas:

The second and third round screening included positive controls adaptedfrom Mohand-Saïd et al. (1998). Five weeks aged mouse 5C57BL/6@N (wildtype) and C3H/He@N (rdl) were sacrificed and enucleated. Two retinaswere dissected and incubated in 24 hours at 37° C. in 5% CO₂ in 1.5 mlof CDCM in 12 wells plates. Conditioned media were recovered andconcentrated by a factor 40 by ultra-filtration on Vivaspin (Sartorius,cutting point 10 kDa). Conditioned media were frizzed in liquid nitrogenand stored in aliquots at −20° C. before used. The day of use,conditioned media were thawed on ice, diluted 10 times in CDCM andsterilized by filtration on 0.22 μm filter (Acrodisk 13, GelmanSciences).

Functional Assay, Live/Dead Assay:

The functional assay is based on the number of chicken retinal cellsalive after 7 days incubation in vitro. We used Live/Dead assay kit(Molecular probes, Eugene, USA) that is based on the use of twofluorogenic dyes (Calcein AM and Ethidium dimer) that stain live anddead cells respectively. A cell that is alive processes a metabolicactivity (here an esterase activity) that coverts the substrate (CalceinAM) in its fluorescent product emitting at 520 nm. The membranepermeability of a dead cell is altered and permits the DNA staining ofthe nucleus by Ethidium dimmer emitting at 635 nm. A cell is alive:emitting at 520 nm after excitation at 485 nM, or dead: emitting at 635nm after excitation at 520 nm. Using epifluorescence microscopy, the twotypes of fluorescent cells can be visualized separately. After 7 days invitro, cells were incubated for 30 minutes at room temperature in thedark with 2.7 μM Calcein-AM and 0.3 mM Ethidium dimer.

Image Acquisition:

Briefly, image acquisition consisted of autofocussing each well,automatic cell counting in two fluorescence's followed by processing ofthe raw data using specialist software e.g. Metamorph (Universal ImagingCorporation, West Chester, USA) to obtain digitalised pictures of eachwell of the plate. We used a inverted microscope (Nikon TE 200) equippedwith a mercury epifluorescent lamp with two excitation filters 485 and520 nm, two emission filters 520 and 635 nm, an object (×10), a computerdriven motorized platine (Multicontrol 2000, Martzauzer and a CCD camera(Cohu).To record the plate, it is positioned on the motorised platine, and thefocus done manually on the first well and this plane is recorded (zorigin). The threshold of the image dead and live is set from the firstwell. The centre of the first well is adjusted using white light byaligning manually the bottom of the first well to the bottom of theimage on the computer monitor, then by aligning the extreme right of thefirst well to the right of the image on the computer screen and torecord the two positions. The centre of the first well is calculated andgives the position of the centre of each well of the plate. We havenoticed in the development process that there is a slight higher densityof cells at the edge of the well and excluded the edge from theacquisitions. It is important that the image from each well be centredperfectly in order to avoid any misleading results. When set-up, thefirst scan of the plate executes a recording of dead cells. The deadcells density is the less variable under these conditions. Theapplication executes an auto focusing by taking images at differentfocal plans and choosing the brightest one, the right focus. This zposition is stored and platine executes programmed movements in the xand y axes taking a total of 4 images that when reconstituted in oneimage represents ⅔ of the surface of the well. A pile of images from thefocus plans is stored for control. The platine executes an auto focusingand four acquisitions for each well of the plate starting by wells A1 toA12, then B12 to B1, C1 to C12 etc. . . . At the end, the platine movesoutside the plate in order to overexpose the last well (H1). Thescanning of dead cells takes 30 minutes. The second scan (live cells) isexecuted after switching the filter. This second scan is using therecorded z positions of each well from the dead scan. Four images aretaken from each well as for dead cells. At the end of the second scan(22 minutes) dead and live reconstituted images are stored in a filethat is named automatically with the date of the day. Cells numbers(dead and line) are counted automatically with pre-set morphometricparameters (average) and displayed on the computer monitor in order tocheck if the experiment is correct. It is important to check on a dailybasis that the number of cells alive is not too high. We have observedthat if plated at too important density, the chicken retinal cellssurvived longer most likely by producing their own survival factor. Wescreened cells in the absence of this effect. Before scanning the secondplate (same experiment with twice density of cells plated), we add an aat the end of the name of the log file from the first plate. Images ofeach experiment were stored on CD-rom. We have generated a library ofmore than 250 CD-rom.

Cells Counting and Selection of the Pools:

The cells numbers (live and dead) were counted using images of eachexperiment stored on CD-rom. The log file from an experiment was firstloaded on a computer (counting off-line) and opened using Metamorphsoftware. In a first step, the images corresponding to the 14 wells C(conditioned media from Cos-1 cells transfected with the empty vector)are opened. After adjusting the threshold of the image, the commandIntegrated Morphometry Analysis is used to measure the distribution ofthe areas between 10 and 250 of the object (number of live cells foreach total area) for these control wells. The distribution follows aGaussian curve with the maximal number of objects corresponding to anisolated cell. This standard value (SV) is then used to calculate thevalue of the area above which an object will be counted as two cells(standard object cut, SOC) through our empirical function: SOC=29/20.74SV. The SOC value of each individual plate is used to count the numberof live cells of the plate. These numbers are then transferred into aexcel table.For the first round of screening, the value were plotted for each poolas fold difference (increase or decrease live cells numbers) versus theaverage of the 14 control wells+the standard deviation. In order toredress the variations coming from the position difference within theplate, we calculated the average of the fold difference individuallybetween the 80 wells corresponding to positions where the pools aretested and the 14 wells control for 200 independent plates. In average,the differences observed are due only to position difference and cellsnumbers were corrected with this coefficient. In order to discriminatein more stringent way the pools to be selected, the live cells countswere plotted as fold difference versus as control all values notexceeding 1.3 but exceeding 0.4. In this manner, all pools were folddifference versus the 14 wells corresponding to empty vector are in theinterval of 0.4 to 1.3 are considered as having no effect and used ascontrol. After correction, the fold difference versus control of the twoplates was multiplied and the result sorted by decreasing folddifference. The pools on the top of that list were further checked byvisual inspection of the graph corresponding to the 20 pools of theexperiment (both plates) and of the images of live and dead cells toavoid to screen misleading pools.For the second and third round, the plates screening for sub-poolsinclude additional control. We prepared conditioned media from retinaexplants of 5 weeks of age. We selected experiments where positiveeffects were recorded for retina explants from C57BL/6@N and dismissedthe others. The results were plotted as fold difference versus the 14control wells, no recalculation of the control was made. The folddifference versus control of the two plates was multiplied and theresults were sorted by decreasing fold difference for the 16 sub-pools.

Isolated cDNA were sequenced using T7 primer (5′GTAATACGACTCACTATAGGGC3′ (SEQ ID NO: 31)) on a capillary sequencer (CEQ2000, Beckman Coulter).DNA sequence was compared to databases using Basic Local AlignmentSearch Tool (BLAST).

Identification of Rdcvf2 and Human Homologues:

Using the Rdcvf1 sequence (the nucleotide sequence encoding thepolypeptides set forth in SEQ ID NO:2 or SEQ ID NO:4) and BLAST,homologous murine and human polypeptides were identified (FIG. 8). ESTclones with homology to mouse RdCVF2 (GenBank Accession No: bc016199)were identified: GenBank Accession Nos: be552141, bi517442, bg707818,bi603812, ai433287, be088414, bg297383, bg297304 (see also FIG. 12). ESTclones with homology to mouse Rdcvf1 (SEQ ID NO:1) were identified:GenBank Accession Nos: bg299078, ai716631, bg294111, be108041, bg395178(see also FIG. 13).

Real-Time RT-PCR Analysis of Rdevf1 Expression

The retinal expression of Rdcvf1 by mouse C57BL/6@N and C3H/He@N aged aswell as congenic C3H (+/+ and rd/rd) of 5 weeks is studied usingreal-time RT-PCR on a lightcycler (Roche) with sybergreen PCR kit(Roche). cDNAs are produced by priming with a random hexameroligonucleotide (pdN6, Amersham), M-MLV reverse transcriptase(superscript II, Life Technologies) and total RNA from mouse retinaprepared as in 1). cDNAs are normalized using a ubiquitous messengerGlucose-6-Phosphate DeHydrogenase (G6PDH). 0.2 μl of first strand cDNAssynthesis (an equivalent of 10 ng of total RNA) is amplified with 2 μMof the oligonucleotides of SEQ ID NO:24 and SEQ ID NO:25 in triple in atotal volume of 25 μl using the following program: 30 seconds at 95° C.,and 35 cycles of a sequence (1 second at 95° C., 18 seconds at 55° C.,10 seconds at 72° C.). The analysis (FIG. 16) shows that Rdcvf1expression decreases after rod degeneration in the rdl mouse (C3H/He@N).Rdcvf1 was also shown to be directly expressed by photoreceptors byreal-time RT-PCR using RNA prepared from outer layer of the retina byvibratome sectionning.

Products were checked by agarose gel electrophoresis. Similar resultsare obtained with another pair of Rdcvf1 specific primers. As a positivecontrol, the expression of rod arrestin (Ass no M24086) is monitored inthe same conditions with primers (5′CTATTACGTCAAGCCTGTAGCC 3′ (SEQ IDNO: 32) and 5′CATCCTCATCTTTCTTCCCTTC 3′ (SEQ ID NO: 33)).

Confirmation that Rdcvf1 is the cone protective factor can be obtainedby adding a suitable amount of Rdcvf1 to a retinal explant of 5 week oldrdl mouse (C3H/He@N). What is a suitable amount can be arrived at bysome initial titration experiments. Compare with appropriate controlsafter 7 days cone survival will be increased.

RT-PCR Analysis of Rdcvf2

RT-PCR for Rdcvf2 expression was carried out using primers5′GCCAGCGTTTTCTGCCTTTTAC 3′ (SEQ ID NO: 34) and 5′AAGCCCTGCCTGCTCTAACATC3′ (SEQ ID NO: 35).

The analysis shows that RdCVF2 is expressed in a rod-dependent mannerand that Rdcvf2 expression is not restricted to the retina, but thatalso other neuronal cells express Rdcvf2 (FIG. 17), whereas theexpression of Rdcvf1 seems to be restricted to retinal cells.

Live/Death Assays of Rdcvf1 or Rdcvf2

COS-1 cells are transfected with a suitable expression vector carryingthe Rdcvf1 or Rdcvf2 under control of a inducible promoter. Controlcells are transfected with the empty vector. The cells are incubated fora suitable period of time upon induction of Rdcvf1 or Rdcvf2 expression.Subsequently, the number of surviving cone cells incubated withconditioned media from COS-1 cells transfected with Rdcvf1 or Rdcvf2 andthe number of surviving control cells are counted according to the abovedescribed method. The cells expressing Rdcvf1 or Rdcvf2 show asignificantly higher amount of surviving cells.

Rod Specific Factor

Real-time RT-PCR analysis, carried out under standard conditions, of theexpression of rod arrestin (control) and Rdcvf1 in 5 weeks retinalexplants from C57BL/6@N 5 weeks and C3H/HE@N, using the primers:

SEQ ID NO: 24: 5′ TCTATGTGTCCCAGGACCCTACAG 3′ SEQ ID NO: 25:5 TTTATGCACAAGTAGTACCAGGACAG 3′demonstrates that RdCVF1 is expressed only in the presence of rods(Rod-derivedCVF1).

Production of Polyclonal Antibodies:

Polyclonal antibodies are prepared by injecting aGluthation-S-Transferase (GST) purified fusion protein (GST-Rdcvf1) aswell as from mouse RdCVF1 peptide sequence amino acids 11 to 32 from SEQID NO₂ (Ab n° 2) and peptide sequence amino acids 79 to 96 from SEQ IDNO₂ (Ab n° 3) into rabbit. The fusion construct pGST-Rdcvf1 is preparedby amplification with oligonucleotides of SEQ ID NO:26 and SEQ ID NO:27using peDNA-Rdcvf1 as template under standard conditions. The OpenReading Frame (ORF) of Rdcvf1 is cloned in frame into pGex2TK(Pharmacia) between the BamHI and EcoRI restriction sites, andtransformed into E. coli [BL21 (DE3) pLysS, Promega] by standardprocedure. A single colony is grown in 3 litres of LB liquid media with100 μg/ml ampicillin at 30° C. and protein production is induced byaddition of 1 μg/ml isopropylthio-β-D-galactoside (IPTG) and continuedfor 5 hours at 30° C. Cells are harvested, lysed be sonication andpurified onto gluthation sepharose under standard protocol. The fusionprotein is eluted with 10 mM reduced gluthation at room temperature. TheEluted protein is dialyzed into PBS before injection to rabbits. Proteinpurity is monitored by polyacrylamide gel electrophoresis. Two rabbitsare immunized by intradermic injection at 80 sites of 100 μg of purifiedGST-Rdcvf1. Serum is harvested after 8 weeks.

What is claimed is:
 1. A pharmaceutical composition comprising: a) anexpression vector comprising a nucleic acid, wherein the nucleic acidcomprises a nucleotide sequence encoding a Rod-Derived Cone ViabilityFactor 1 (RDCVF1) or a Rod-Derived Cone Viability Factor 2 (RDCVF2)polypeptide and wherein the nucleotide sequence is operably linked to anexpression control sequence, and b) a pharmaceutically acceptablecarrier.
 2. The pharmaceutical composition of claim 1, wherein theexpression vector is a viral vector.
 3. The pharmaceutical compositionof claim 2, wherein the viral vector is an adeno-associated virusvector.
 4. The pharmaceutical composition of claim 2, wherein the viralvector is an adenovirus vector.
 5. The pharmaceutical composition ofclaim 1, wherein the expression control sequence is an induciblepromoter.
 6. The pharmaceutical composition of claim 1, wherein theexpression control sequence is a constitutive promoter.
 7. Thepharmaceutical composition of claim 1, wherein the expression controlsequence is a tissue specific promoter.
 8. The pharmaceuticalcomposition of claim 1, wherein the polypeptide is a human RDCVF1polypeptide.
 9. The pharmaceutical composition of claim 8, wherein thepolypeptide is a long form of the human RDCVF1 polypeptide.
 10. Thepharmaceutical composition of claim 8, wherein the polypeptide is ashort form of the human RDCVF1 polypeptide.
 11. The pharmaceuticalcomposition of claim 1, wherein the polypeptide is a human RDCVF2polypeptide.
 12. The pharmaceutical composition of claim 11, wherein thepolypeptide is a long form of the human RDCVF2 polypeptide.
 13. Thepharmaceutical composition of claim 11, wherein the polypeptide is ashort form of the human RDCVF2 polypeptide.
 14. The pharmaceuticalcomposition of claim 1, wherein the polypeptide comprises the amino acidsequence of SEQ ID NO:6.
 15. The pharmaceutical composition of claim 1,wherein the polypeptide comprises the amino acid sequence of SEQ IDNO:8.
 16. The pharmaceutical composition of claim 1, wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:14.